We have examined the respective roles in EAU of the Th1 and the Th17 effector pathways in uveitis by using monoclonal antibodies and mutant mice deficient in various components of these pathways. We found that IL-23 and not IL-12 were necessary for development of EAU. Depending on the model, autoimmunity to retina could be either Th17 or Th1 driven, to the point of mediating disease as a standalone effector. That the conditions that determine which effector type will predominate appear to include the innate context in which Ag exposure occurs. The conditions that lead to a Th17 driven disease are strong TLR stimulation (CFA) and a diversity of APC, but when disease is induced in the context of a more focused stimulation, such as in vitro matured Ag pulsed dendritic cells, Th1 is dominant. These data shed light on the heterogeneity of human uveitis and may impact on therapy by suggesting that either the Th17 or the Th1 pathways may be appropriate targets in different types of human uveitis (1).[unreadable] [unreadable] We found that mouse as well as human NKT cells make a very rapid IL-17 response upon ligation of the T cell receptor (TCR) and/or the IL-23 Receptor. The two pathways are independent and additive. NKT have constitutive expression of IL-23R and ROR&#947;t, and their IL-17 response is independent of IL-6 and appears independent of IL-21. All known NKT subtypes (type I, II and III, defined by response to &#945;&#8722;GalCer and dependence on CD1d) make IL-17. However, unlike IFN-gamma and IL-4, IL-17 is made only by the NK1.1-negative subset. The relevance, if any, of this innate IL-17 response to autoimmunity and to EAU, is unknown at this time (2). [unreadable] [unreadable] The role of NKT-produced IFN-g appears can, on the other hand, be protective. Triggering NKT in vivo by treatment with alpha-GalCer at the time of uveitogenic immunization inhibits development of EAU. This was causally related to inhibition down the line of the adaptive Th1 and Th17 responses. This contrasts with reports in some other autoimmune disease models, which connected the protective effect of NKT to induction of IL-4 and Th2 polarization. These findings are doubly interesting in view of the reported role of NKT as participants in the process of eye-induced tolerance, contributing to ocular immune privilege, and suggest that NKT cells can protect from EAU at more than one level. (3).[unreadable] [unreadable] Sequestration of retinal Ags behind the blood-retinal barrier hinders peripheral tolerance and renders the individual susceptible to EAU. Exposure to IRBP in the periphery under tolerogenic conditions, by "vaccination for tolerance" can prevent and to a lesser extent can reverse EAU. In the previous review year we used naked DNA vaccination. In this review year we studied the tolerogenic effect of inhibitory peptide analogs administered in incomplete Freund's adjuvant. Characterization of the mechanisms revealed involvement of T regulatory cells (Tregs) and Th2 skewing. Tolerogenic vaccination, or transfer of in vitro expanded Treg cells, may offer a new approach to Ag-specific therapy of uveitis. (4)[unreadable] [unreadable] L-10 is a known regulatory cytokine that our previous studies connected to resolution of EAU as well as to genetic resistance to the disease. We now show that transgenic IL-10 expression in macrophages, and to a lesser extent in T cells, protects from EAU at several levels. It inhibits priming of new uveitogenic effector T cells from na&#239;ve precursors, and also inhibits the function of already mature effectors. These data suggest that increasing endogenous expression of IL-10 by gene transfer could be developed as a therapeutic approach. (5).[unreadable] [unreadable] We were able to identify several new pathogenic peptides for the C57BL/6 (H-2b) and B10.RIII (H-2r) strains. Each peptide elicited responses of both CD4 and CD8 cells. Some of these represent specificities recognized only by IRBP KO but not WT mice (i.e., become reduced or eliminated through central tolerance in WT mice of the respective strains). C57BL/6 mice recognized more KO-only epitopes, suggesting more extensive repertoire culling in this strain. These findings will be useful for immunological studies that require knowledge of multiple epitopes and will help to understand the pathogenic potential of IRBP as an auto-antigen (6). [unreadable] [unreadable] Within the scope of our studies on genetic susceptibility to uveitis in the rat EAU model using an F2 cross of EAU-susceptible and resistant strains, we have defined a number of genes that may be involved in susceptibility. The methods use combined approaches of classical genetics, microarray analysis of expressed genes and in silico analysis (the latter in collaboration with Roche Pharmaceuticals). Some susceptibility regions are unique, and could be mapped to specific genes using microarray and in silico approaches, and some are shared with other autoimmune and non-autoimmune diseases, among them EAE, arthritis and type 2 diabetes. This indicates that uveitis shares common pathways with other autoimmune and inflammatory diseases, and may suggest use of common therapeutic approaches (7, 8).[unreadable] [unreadable] Immunological responses to S-Ag have been implicated in human uveitis, however, direct study of uveitogenic epitopes in humans is not possible. We have established a "humanized" EAU model in HLA Tg mice. Using bioinformatic methods for epitope prediction we are studying recognition and pathogenicity of S-Ag epitopes, and in some cases identifying their core sequences We have identified permissive HLA-DR3(0301), HLA-DR4(0402), HLA-DQ8 and non-permissive HLA-DR4(0401), HLA-DR2 (*1501, *1502, and *1503) alleles of the HLA-DR and DQ genes, and characterized some of the allele-specific uveitogenic epitopes of human S-Ag. Importantly, the sequences of these epitopes overlap with peptide M and peptide N of S-Ag, and the S-Ag crossreactive B27PD peptide derived from HLA-B27 molecule, that were reported to be recognized by lymphocytes of uveitis patients. We are currently developing MHC tetramers to study lymphocytes with T cell receptors specific to these epitopes in the HLA Tg mice and in uveitis patients. These data will provide new insights into the pathogenesis if uveitis and may help to develop therapeutic strategies tailored to the specific HLA haplotype of the patient. (Mattapallil et al., manuscript in preparation).[unreadable] [unreadable] We continue to examine the effect of retinal glial M&#252;ller cells on uveitogenic T lymphocytes. We have previously shown that retinal glial Muller cells inhibit proliferation of T cells in an Ag-independent fashion by a contact dependent mechanism. Using primary cultures of murine Muller cells and genetically manipulated mice, we have identified thrombospondin-1 and TGF-beta as partly responsible for the suppressive effect of Muller cells. T cells that have interacted with Muller cells downregulate any preexisting expression of IL-2 receptor and FoxP3. We are currently studying other functional consequences on the T cells that came in contact with M&#252;ller cells. (Cortes et al., manuscript in preparation).